TY - JOUR
AU1 - Brunetti,, Cecilia
AU2 - Savi,, Tadeja
AU3 - Nardini,, Andrea
AU4 - Loreto,, Francesco
AU5 - Gori,, Antonella
AU6 - Centritto,, Mauro
AB - Abstract Drought compromises plant's ability to replace transpired water vapor with water absorbed from the soil, leading to extensive xylem dysfunction and causing plant desiccation and death. Short-term plant responses to drought rely on stomatal closure, and on the plant's ability to recover hydraulic functioning after drought relief. We hypothesize a key role for abscisic acid (ABA) not only in the control of stomatal aperture, but also in hydraulic recovery. Young plants of Populus nigra L. were used to investigate possible relationships among ABA, non-structural carbohydrates (NSC) and xylem hydraulic function under drought and after re-watering. In Populus nigra L. plants subjected to drought, water transport efficiency and hydraulic recovery after re-watering were monitored by measuring the percentage loss of hydraulic conductivity (PLC) and stem specific hydraulic conductivity (Kstem). In the same plants ABA and NSC were quantified in wood and bark. Drought severely reduced stomatal conductance (gL) and markedly increased the PLC. Leaf and stem water potential, and stem hydraulic efficiency fully recovered within 24 h after re-watering, but gL values remained low. After re-watering, we found significant correlations between changes in ABA content and hexoses concentration both in wood and bark. Our findings suggest a role for ABA in the regulation of stem carbohydrate metabolism and starch mobilization upon drought relief, possibly promoting the restoration of xylem transport capacity. Introduction Forestry is one of the economic sectors most vulnerable to ongoing climate change. A pervasive consequence of the current warming trend is an increasing frequency and intensity of drought periods, representing a major threat for trees (Allen et al. 2010, Centritto et al. 2011a). Drought-induced damage to trees ranges from reduced plant growth an d productivity to increased mortality rates, and it is generally associated with a substantial loss of xylem hydraulic function (McDowell et al. 2008, Adams et al. 2017). The term ‘hydraulic dysfunction’ refers to the impairment in functionality of the xylem network caused by embolism (Tyree and Sperry 1989, Mitchell et al. 2013). Embolism is the physical process of formation and propagation of an air phase into a functioning xylem conduit containing water under tension (Zwieniecki and Secchi 2015, Rodriguez-Dominguez et al. 2018). This phenomenon occurs not only in the stem xylem vessels but also in the roots and in the leaves. Indeed, recent studies have detected both embolism formation and tissue shrinkage in roots of droughted plants (Pratt et al. 2015, Cuneo et al. 2016); previous studies have also confirmed embolism formation in the leaves (Nardini et al. 2001), but it can be considered as a rare event associated with severe water stress (Brodribb et al. 2016). Embolism build-up decreases hydraulic conductance in stems (Kstem), leaves (Kleaf) and roots (Kroot), thus causing partial or total stomatal closure and an unavoidable reduction of photosynthesis (Tyree and Sperry 1989, Brodribb 2009). However, further evidences suggest that, in some species exposed to water stress, stomatal closure pre-empts xylem embolism in the leaf, thus acting as a safety mechanism (Brodribb et al. 2016, Hochberg et al. 2017). The vulnerability to embolism is species-specific and tissue-specific, with some trees displaying marked resistance to hydraulic dysfunction, while others can lose Kstem at relatively high xylem water potential (Ψx) (Choat et al. 2012). Interestingly, most angiosperm trees can survive with losses of xylem hydraulic conductance close to 50%, although this is unlikely to be the case for current-year xylem (Choat et al. 2012, Brodersen and McElrone 2013). However, some studies suggest that in some species reversal of embolism and hydraulic recovery can occur upon drought relief (Tyree and Sperry 1989, Brodersen et al. 2018, Klein et al. 2018, Love and Sperry 2018). Although controversial, the mechanism of refilling of gas-filled conduits with water, leading to restoration of the hydraulic pathway from roots to shoots, has been detected in several species using both classical hydraulic techniques and new in vivo imaging analyses (Brodersen et al. 2010, Secchi and Zwieniecki 2010, Nardini et al. 2011, Zwieniecki et al. 2013, Mayr et al. 2014, Trifilò et al. 2015, Ooeda et al. 2017). Xylem hydraulic recovery has been reported to occur over time intervals ranging between 10 min to more than 15 h (Tyree and Sperry 1989, Brodersen et al. 2010, Trifilò et al. 2014, Klein et al. 2018). The occurrence and magnitude of recovery depends on several factors including plant species (Brodersen and McElrone 2013, Trifilò et al. 2015), plant water status prior to refilling (Hacke and Sperry 2003), occurrence of root pressure, non-structural carbohydrate (NSC) at content the end of drought (Savi et al. 2016a, Trifilò et al. 2017, Tomasella et al. 2017), size of the conduits (Brodersen et al. 2010) and hydrostatic pressure in functioning conduits in the xylem network (Tyree and Sperry 1989, Brodersen et al. 2013). Several studies support the idea that the hydraulic recovery process in xylem is driven by osmotic pressures generated locally (Salleo et al. 2009, Secchi and Zwieniecki 2012, Secchi and Zwieniecki 2016). This assumption is based on the observation that temporal patterns of embolism repair are paralleled by changes in carbon partitioning between starch and NSC in the wood parenchyma (Bucci et al. 2003, Salleo et al. 2004, Salleo et al. 2009, Zwieniechi & Secchi 2015). Moreover, molecular studies have revealed changes in α-β amylases activity in the stem (Secchi and Zwieniecki 2011, Perrone et al. 2012), a reduction in pH values of xylem sap (Secchi and Zwieniecki 2016) and up-regulation of several genes (sucrose transporters, PIPs and TIPs acquaporins) upon embolism recovery (Laur and Hacke 2014, Mayr et al. 2014, Secchi et al. 2017). During recovery, the build-up of sugars and possibly other organic or inorganic solutes is thought to generate a local increase in the osmotic pressure that would provide the driving force for water entry in refilling conduits (Bucci et al. 2003, Zwieniecki and Hlobrook 2009, Secchi and Zwieniecki 2011, Stroock et al. 2014). Although xylem and phloem are often studied separately as distinct and isolated tissues, water transport in the xylem and translocation of sugars in the phloem are two partially interdependent mechanisms, and their fine tuning determines plant performance (Nardini et al. 2011, Spicer 2014). Water transfer from the phloem to the xylem has been observed in both well-watered (Pfautsch et al. 2015) and water-stressed conditions (Tyree et al. 1999). In addition, the involvement of the phloem in xylem hydraulic recovery has been postulated (Nardini et al. 2017). Abscisic acid (ABA) is an important plant hormone, well known for its large and rapid increase in concentration when plants face environmental stresses. Abscisic acid may affect plant growth, productivity and survival during drought. Previous studies have suggested a role for ABA in sugar mobilization in different plant tissues (Pan et al. 2005, Kempa et al. 2008, Tattini et al. 2014). For instance, ABA-induced starch degradation was observed in leaves and roots of Arabidopsis thaliana by Kempa et al. (2008). In addition, a complex re-adjustment of carbohydrate metabolism paralleled by rising ABA levels was reported in tobacco plants exposed to drought (Tattini et al. 2014). In this context, it has been proven that ABA modulates carbohydrate metabolism by inducing the activation of different enzymes, such as β-amylase and vacuolar invertase (Pelleschi et al.1999, Trouverie et al. 2004, Pan et al. 2005, Thalmann et al. 2016), which catalyze the irreversible conversion of starch into soluble sugars. Although the role of ABA on stomatal closure has been extensively investigated in many studies (McAinsh et al. 1990, Davies et al. 2002, Munemasa et al. 2015, Takahashi et al. 2018), the possible function of this hormone in stem NSC metabolism for the generation of osmotic forces during post-drought hydraulic recovery has received little attention so far (Secchi et al. 2013). In the present study, we report results from an experiment aimed at verifying a possible relationship between ABA and NSC metabolism during embolism formation and recovery in Populus nigra L., a species where xylem refilling has been previously reported (Secchi and Zwieniecki 2012). Although P. nigra is a drought sensitive and high-water demanding species, in the last years it has gained interest as a woody crop for biomass production in short rotation forestry systems (Garavillon-Tournayre et al. 2018) with multiple industrial applications. Thus, a better knowledge of the capability of P. nigra to recover from xylem embolism could be important to predict its resilience to future global change-type droughts. In particular, we used P. nigra as a model species to investigate: i) how the stem NSC pools are mobilized under drought and subsequent re-watering; and ii) the different dynamics of ABA and NSC in two separate tissues, bark and wood, thus dissecting the possible role of this hormone in the hydraulic recovery. Materials and methods Plant material and drought-stress treatment Two-year-old cuttings of Populus nigra L. (height 40–45 cm and diameter 1–1.2 cm) were grown in 20 dm3 pots filled with sandy soil, with a saturated water content and an available water content of 0.39 g g−1 and 0.37 g g−1, respectively. Before the onset of the experiment, plants were regularly watered and fertilized with half-strength Hoagland. The water stress experiment was performed in the glasshouse of the University of Trieste in August 2016, when plants were randomly assigned to two experimental treatments, i.e., well-watered (WW, 15 plants) and water-stressed (WS, 15 plants). To assign plants to each category, preliminary measurements of stomatal conductance to water vapor (gL), maximum photochemical efficiency of photosystem II (PSII) (Fv/Fm) and leaf water potential (ΨL) were done to exclude significant differences among plants (P > 0.05, data not shown). The WW plants were irrigated daily to pot capacity during the experimental period (Ψsoil = 0 MPa), while irrigation was withheld to WS plants for 20 days. At the end of the drought treatment (Ψsoil = −1.56 MPa), five WS plants were re-irrigated to pot capacity (RW) the evening before measurements (see following sections), checking that soil water potential was zero the following morning. Soil water potential was measured on three soil samples per pot taken on the surface (−5/10 cm from the top of the soil) between 12:00 and 14:00 h (solar time). The soil water potential was measured in the laboratory with a pre-calibrated hygrometer (WP-4, Decagon Devices Inc., Pullman, WA, USA). During the water stress treatment, mean air temperature and relative humidity were monitored using an EasyLog-USB-2 (Lascar Electronics Inc., Salisbury, UK), while the photosynthetic photon flux density (PPFD) was measured using a type sensor quantum/photometer/radiometer (model HD-9021, Delta Ohm s.r.l., Padova, Italy). Measurements of plants water status and photosynthetic efficiency To assess the impact of drought stress and re-watering on plant functioning, water status and chlorophyll-a fluorescence were measured at the end of the water stress period and after rewatering. Stomatal conductance to water vapor was measured between 12:00 and 14:00 h (solar time) with a pre-calibrated steady state porometer (SC-1, Decagon Devices Inc.). After gL measurements, Fv/Fm was estimated with a portable fluorometer (Handy Pea, Hansatech, Norfolk, UK) (Maxwell and Johnson 2000). The selected leaves were darkened 20 min before measurements. Leaves were then sampled and wrapped in cling film, and their midday leaf water potential (ΨLM) was measured with a pressure chamber (mod. 1505D, PMS Instruments, Albany, OR, USA). Xylem water potential (ΨX) was also measured on leaves which were wrapped in cling film and aluminium foil 1 h earlier, to prevent transpiration and overheating. Leaves for measurement of ΨX were chosen close to the part of the stem used for PLC measures. On the same measurement dates, pre-dawn leaf water potential (ΨLP) was measured at 5:00 h. All measurements were performed on at least two leaves per individual plant. Measurements of stem hydraulic conductivity and drought-induced hydraulic impairment In order to assess the impact of the drought treatment on water transport efficiency of plants (WW vs WS) and to monitor eventual hydraulic recovery after re-watering (WS vs RW), the percentage loss of hydraulic conductivity (PLC), stem specific hydraulic conductivity (Kstem) and stem maximum hydraulic conductivity (Kmax) were measured at the end of water stress period and after rewatering. At the peak of the water stress treatment and about 20 h after re-irrigation, the main stem of WW, WS and RW plants was cut at the root collar while immersed in water. To further avoid spurious formation of embolism in samples excised under tension (Wheeler et al. 2013), the stem was progressively trimmed at both sides with a razor blade, debarked and two final 4 cm long segments (corresponding to the central part of the stem) were connected to the Xyl’Em apparatus (Xylem Embolism Meter, Bronkhorst, Montigny-les-Cormeilles, France). The maximum vessel length of Populus nigra is about 20 cm (Pivovaroff et al. 2016), but this value was likely much lower for our small plants, and in any case it was not expected to affect the hydraulic measurements considering the precautions adopted to relax xylem tension. The initial hydraulic conductance (Ki) of each sample was measured during perfusion with a filtered poly-ionic solution (enriched with 10 mM KCl) at a pressure of about 6 kPa (Nardini et al. 2007). Samples were then flushed at high pressure (0.2 MPa) for 5 min. After embolism removal, hydraulic conductance was re-measured (Kf) as described above, and PLC calculated as 100 × (1 − Ki/Kf). Stem-specific hydraulic conductivity (Kstem) was calculated by multiplying Ki per sample length and dividing per transverse xylem area. Stem maximum hydraulic conductivity (Kmax) was calculated by multiplying Kf per sample length and dividing per transverse xylem area. The PLC, Kstem and Kmax measured in the two sub-samples of each individual were averaged. All hydraulic measurements were performed between 10:00 and 14:00 h (solar time) on the same plants previously used for gL, Fv/Fm and Ψ measurements. Measurements of leaf osmotic potential Osmotic potential of leaves (ΨΠ) was measured at the end of the water stress period and after rewatering to detect eventual osmoregulation processes triggered by drought stress. Immediately after stem sampling for hydraulic analyses, five leaves from each individual were detached, cut in small pieces, wrapped in cling film and frozen in liquid nitrogen to break cells and release cell sap. Samples were then mashed, and osmotic potential was measured with a pre-calibrated hygrometer (WP-4, Decagon Devices Inc., Savi et al. 2016b). Measurements of free-ABA and its metabolites/catabolites Xylem sap for quantification of free-ABA and ABA glucoside ester (ABA-GE) was collected from the same plants used to measure PLC, using the procedure described in Secchi and Zwieniecki (2012). Immediately after cutting the stem, leaves were removed and after detaching the bark from the first 5-cm piece of stem, the stem was attached to a small vacuum chamber. After generation of a vacuum (0.03 MPa absolute pressure), small stem segments were progressively cut from the top, allowing xylem sap to be sucked out of the stem and collected in an Eppendorf tube. The collected sap was frozen and kept at −80 °C until analysis. In the small pieces of stem previously cut, the bark (including cambium) was separated from wood with a razor blade and both portions were frozen in liquid nitrogen to stop enzymatic activity and then lyophilized. Abscisic acid, both in free (ABA) and conjugated (ABA-GE) form, and its catabolites (phaseic (PA) and dihydrophaseic (DPA) acids) were extracted and quantified from bark, wood and xylem sap as reported in López-Carbonell et al. (2009), with little modification. In detail, 60 mg of lyophilized tissue were ground in liquid nitrogen and mixed with 50 ng of d6-ABA, d5-ABA-GE, d3-PA and d3-DPA (National Research Council of Canada), then extracted with 3 × 1 ml pH 2.5 CH3OH/H2O (50:50; v:v) at 4 °C for 30 min. The supernatant was defatted with 3 × 3 ml of n-hexane, purified using Sep-Pak C18 cartridges (Waters, Milford, MA, USA) and eluted with 1 ml of ethylacetate. The eluate was dried under nitrogen, rinsed with 500 μl pH 2.5 CH3OH/H2O (50:50) and then injected (3 μl aliquots) in a LC–DAD-MS/MS system consisting of a Shimadzu Nexera HPLC and a Shimadzu LCMS-8030 quadrupole mass spectrometer, operating in electrospray ionization (ESI) mode (Shimadzu, Kyoto, Japan). The eluting phases consisted of H2O (added with 0.1% of HCOOH, solvent A) and CH3CN/CH3OH (1:1, v:v, added with 0.1% of HCOOH, solvent B). The analysis was performed in negative ion mode, using a 3 × 100 mm Poroshell 120 SB C18 column (2.7 μm, 100 × 4.6 mm, Agilent Technologies, Palo Alto, CA, USA) and eluting a 18 min-run from 95% solvent A to 100% solvent B at a flow rate of 0.3 ml min−1, Quantification was conducted in multiple reaction mode (MRM). For xylem sap analyses, 3 μl of sap were injected after the addition of internal standards. Measurements of carbohydrates and starch Soluble carbohydrates in bark and wood were quantified by HPLC-RI analysis on samples collected at the end of the water stress period and after rewatering, extracting 100 mg of lyophilized tissue with 3 × 5 ml of ethanol/water (75/25). The solvent was reduced to dryness under vacuum and the resulting pellet was rinsed with 2 ml water. The aqueous extract was then purified by solid-liquid extraction through -CH and -SAX pre-packed Bond-Elute cartridges (Varian, Harbor City, CA, USA) and the eluate reduced to dryness under vacuum. Samples were rinsed with ultrapure water and injected in a Series 250 LC binary pump equipped with an LC 30-RI detector (Perkin-Elmer, Shelton, CT, USA). Soluble carbohydrates were separated on a 7.7 × 300 mm Hi-Plex Ca column (Agilent Technologies) maintained at 88 ± 1 °C. Eluent was ultra-pure water at a flow rate of 0.8 ml min−1 during a 20-min run. Individual carbohydrates were identified by comparison of retention times with those of authentic carbohydrate standards (Sigma-Aldrich, Milano, Italy). Starch was quantified as reported in Chow and Landhäusser (2004) on the dry pellet from soluble sugars extraction. The pellet was re-suspended in 1 ml of distilled water and starch digested through an enzyme mixture consisting of 1000 U ml−1 α-amylase and 5 U ml−1 of amyloglucosidase. Glucose was quantified through peroxidase-glucose oxidase/o-dianisidine reagent (Starch Assay Kit, Sigma-Aldrich), reading the absorbance at 525 nm after the addition of sulfuric acid. Anatomical measurements and calculations of theoretical amount of glucose required for PLC recovery Six 5 cm long stem segments sampled from WW plants were placed in distilled water for overnight rehydration. Three stems were used for anatomical analyses, while the other three for stem density measurements. Thin transverse slices were cut using a razor blade and images of cross sections were acquired with a digital camera (Leica DC-300F, Leica Camera, Solms, Germany). The area of the entire wood section (Awood) and the area occupied by xylem vessels (Avessels) were measured with ImageJ (1.46r, NI). The volume occupied by xylem vessels lumina (Vvessels) was estimated as Avessels/Awood and turned out to be 0.11. The fresh volume of three intact (FVbark + wood) and debarked (FVwood) stems was measured with the water displacement method (Hughes 2005), and the bark volume (FVbark) calculated as FVbark + wood–FVwood. Wood and bark samples were weighed (DWwood, DWbark) after drying (24 h at 70 °C) and the relative densities (Dwood, Dbark) were calculated as DWwood/FVwood and DWbark/FVbark, respectively. Taking into account that xylem conduits lumina occupy approximately 11% of wood volume, and that wood and bark density were about 0.37 g cm−3 and 0.41 g cm−3, respectively, we utilized the Van’t Hoff equation to calculate the theoretical amount of glucose (mg g−1 DW) necessary to generate an osmotic potential negative enough to counterbalance the residual negative ΨX following re-watering, thus making possible embolism repair in stems (Nardini et al. 2017). Experimental design and statistical analysis Over the study period, the WW plants maintained stable values of all measured physiological and biochemical parameters. Generalized Linear Models (GLM) were used to highlight differences in physiological parameters among experimental groups measured at the end of the drought treatment/rewatering. The analyses were performed using R software (R i386 3.2.5) through GLM function followed by Tukey’s post hoc tests. Specifically, differences among WW, WS and RW plants were tested via one-way analysis of variance followed by Tukey’s post hoc test. Linear regression analysis was used to determine the relationships between ABA and soluble carbohydrates using Sigmaplot (SPSS, Inc., Chicago, IL, USA). Means are reported ± standard error of the mean (SE). Results During experimental period, air temperatures and relative humidity in the glasshouse averaged 30.5 °C and 50%, while PPFD and vapor pressure deficit (VPD) were about 420 μmol m−2 s−1 and 1.9 kPa, respectively. Under drought, gL significantly decreased (−56%), and did not recover overnight upon re-watering (Figure 1a). The maximum quantum yield of PSII (Fv/Fm) (Figure 1b) declined slightly, but significantly, from 0.8 to 0.76 in WS leaves compared with WW leaves, and totally recovered in RW leaves, indicating that the photochemical efficiency was fully recovered upon rehydration. Figure 1. Open in new tabDownload slide Leaf conductance to water vapor (gL) (A) and maximum quantum yield of PSII (Fv/Fm) (B) measured in control (WW, white bars, n = 11), drought-stressed (WS, black bars, n = 4) and re-irrigated (RW, gray bars, n = 5) plants of Populus nigra. Means ±SE were separated by Tukey’s test and different letters indicate means statistically different with P < 0.05. Figure 1. Open in new tabDownload slide Leaf conductance to water vapor (gL) (A) and maximum quantum yield of PSII (Fv/Fm) (B) measured in control (WW, white bars, n = 11), drought-stressed (WS, black bars, n = 4) and re-irrigated (RW, gray bars, n = 5) plants of Populus nigra. Means ±SE were separated by Tukey’s test and different letters indicate means statistically different with P < 0.05. Water deficit did not produce large effects on water potential parameters (Figure 2). Over the whole study period, plants maintained stable values of ΨLP (ranging between 0 and − 0.2 MPa) without significant differences among the treatments (Figure 2C). Nevertheless, WS plants showed lower, but not significantly different, values of ΨLM (~−30%, Figure 2a) and ΨX (~−30%, Figure 2b) than fully irrigated plants (WW and RW). These differences were statistically significant when comparing WS plants and RW plants. Leaf osmotic potential (Ψπ, Figure 2D) did not change among treatments. Figure 2. Open in new tabDownload slide Leaf water potential (ΨLM) (A), xylem water potential (ΨX) (B), predawn leaf water potential (ΨLP) (C) and osmotic potential (Ψπ) (D) measured in control (WW, white bars, n = 11), drought-stressed (WS, black bars, n = 4) and re-irrigated (RW, gray bars, n = 5) plants of Populus nigra. Means ± SE were separated by Tukey’s test and different letters indicate means statistically different with P < 0.05. Figure 2. Open in new tabDownload slide Leaf water potential (ΨLM) (A), xylem water potential (ΨX) (B), predawn leaf water potential (ΨLP) (C) and osmotic potential (Ψπ) (D) measured in control (WW, white bars, n = 11), drought-stressed (WS, black bars, n = 4) and re-irrigated (RW, gray bars, n = 5) plants of Populus nigra. Means ± SE were separated by Tukey’s test and different letters indicate means statistically different with P < 0.05. The water stress treatment induced an increase in embolism rates in WS plants compared with WW ones (Figure 3). The PLC was about 41% in WW plants and about 63% in WS plants. Following re-watering, PLC returned to values observed in WW plants. Similar patterns were observed when comparing native values of Kstem and Kmax among treatments (Figure 3B and C). These patterns were reflected in Kstem, which was lower for RW than for WW plants, and lowest for WS (Figure 3b). Although Kmax values showed similar qualitative patterns, differences among treatments were not statistically significant in this case (Figure 3C). Figure 3. Open in new tabDownload slide Percentage loss of hydraulic conductivity (PLC) (A), stem specific hydraulic conductivity (Kstem) (B) and stem maximum hydraulic conductivity (Kmax) (C) measured in control (WW, white bars, n = 11), drought-stressed (WS, black bars, n = 4) and re-irrigated (RW, gray bars, n = 5) plants of Populus nigra. Means ± SE were separated by Tukey’s test and different letters indicate means statistically different with P < 0.05. Figure 3. Open in new tabDownload slide Percentage loss of hydraulic conductivity (PLC) (A), stem specific hydraulic conductivity (Kstem) (B) and stem maximum hydraulic conductivity (Kmax) (C) measured in control (WW, white bars, n = 11), drought-stressed (WS, black bars, n = 4) and re-irrigated (RW, gray bars, n = 5) plants of Populus nigra. Means ± SE were separated by Tukey’s test and different letters indicate means statistically different with P < 0.05. We assessed the variability of ABA and its related metabolites/catabolites accumulation in the stem (i.e., bark, wood and xylem sap) of P. nigra in response to soil drying and re-watering (Figure 4). Free-ABA content increased significantly in the bark and in the wood in RW plants compared with WW and WS, while in the xylem sap the concentration of this hormone increased from 0.15 nmol ml−1 (WW) to 0.50 nmol ml−1 in WS samples and then reached the maximum of 4.30 nmol ml−1 in RW samples. Water stress did not affect ABA-GE content in bark and xylem sap. In wood, the increase of this metabolite was marginally significant in WS plants compared with WW, reaching significantly higher values in RW plants. To investigate the turnover of ABA, the main catabolites phaseic (PA) and dihydro-phaseic acid (DPA), were also analyzed. The highest contents were found in RW samples, where PA increased up to five fold in bark compared with WW samples, while the increase was more than ten-fold in wood and in xylem sap. Dihydro-phaseic acid showed the same trend of accumulation in bark, wood and xylem sap, increasing from WW to WS and from WS to RW. Figure 4. Open in new tabDownload slide Levels of abscisic acid (ABA) (A), abscisic acid-glucose ester (ABA-GE) (B), phaseic acid (PA) (C) and dihydro-phaseic acid (DPA) (D) in control (WW, white bars), drought-stressed (WS, black bars) and re-irrigated (RW, gray bars) plants of Populus nigra. Means ± SE (n = 4) were separated by Tukey’s test and different letters indicate means statistically different with P < 0.05. Figure 4. Open in new tabDownload slide Levels of abscisic acid (ABA) (A), abscisic acid-glucose ester (ABA-GE) (B), phaseic acid (PA) (C) and dihydro-phaseic acid (DPA) (D) in control (WW, white bars), drought-stressed (WS, black bars) and re-irrigated (RW, gray bars) plants of Populus nigra. Means ± SE (n = 4) were separated by Tukey’s test and different letters indicate means statistically different with P < 0.05. Water deficit did not significantly affect NSC in the bark, but increased fructose concentration in the wood (Figure 5). After re-watering, a statistically significant increase in glucose and fructose was recorded both in bark and in wood. In particular, glucose content increased by about 20% in RW plants compared with WW plants. Sucrose content, that represents a minor fraction of soluble carbohydrates in P. nigra stems, showed a significant decline in bark of RW plants (−25%) compared with WW, while in wood it increased by 38% after re-watering. In addition, starch content in wood was not affected by water deficit, but significantly decreased (−34%) in bark samples from WS to RW. Figure 5. Open in new tabDownload slide Levels of glucose (A), fructose (B), sucrose (C) and starch (D) in control (WW, white bars), drought-stressed (WS, black bars) and re-irrigated (RW, gray bars) plants of Populus nigra. Means ± SE (n = 4) were separated by Tukey’s test and different letters indicate means statistically different with P < 0.05. Figure 5. Open in new tabDownload slide Levels of glucose (A), fructose (B), sucrose (C) and starch (D) in control (WW, white bars), drought-stressed (WS, black bars) and re-irrigated (RW, gray bars) plants of Populus nigra. Means ± SE (n = 4) were separated by Tukey’s test and different letters indicate means statistically different with P < 0.05. Statistically significant linear correlations (P ≤ 0.05) between free-ABA contents and soluble NSC were observed both in bark and in wood (Figure 6). By contrast, no correlation emerged between ABA and starch in wood (Figure 7), while a negative statistically significant relationship was found in bark (P = 0.02; Figure 7). The relationships between soluble NSC and free-ABA were positive for all sugars in both tissues (Figure 6), but not for sucrose in bark (negative correlation, Figure 6C). Figure 6. Open in new tabDownload slide Linear regressions showing the relationships between ABA and soluble carbohydrates in bark and wood of Populus nigra control (WW, white circles), drought-stressed (black circles) and re-irrigated (gray circles) plants. The area within the dotted lines indicates the 95% confidence bands of the fitted lines. Figure 6. Open in new tabDownload slide Linear regressions showing the relationships between ABA and soluble carbohydrates in bark and wood of Populus nigra control (WW, white circles), drought-stressed (black circles) and re-irrigated (gray circles) plants. The area within the dotted lines indicates the 95% confidence bands of the fitted lines. Figure 7. Open in new tabDownload slide Linear regressions showing the relationships between ABA and starch in bark and wood of Populus nigra control (WW, white circles), drought-stressed (black circles) and re-irrigated (gray circles) plants. The area within the dotted lines indicates the 95% confidence bands around the fitted lines. Figure 7. Open in new tabDownload slide Linear regressions showing the relationships between ABA and starch in bark and wood of Populus nigra control (WW, white circles), drought-stressed (black circles) and re-irrigated (gray circles) plants. The area within the dotted lines indicates the 95% confidence bands around the fitted lines. Theoretical calculations on a representative stem segment (length 5 cm, diameter 8 mm) suggested that an osmotic potential of at least −0.67 MPa (i.e., 0.1 MPa more negative than ΨX) in the embolized conduits volume of RW plants with a PLC of 63% could be generated by approximately 5.7 mg g−1 DW of free glucose in stem (wood and bark). Discussion Poplars are highly productive trees in temperate latitudes, and are thus cultivated for timber, pulp and fuelwood (Harvey and Van den Driessche 1997; Rood et al. 2003; Fichot et al. 2009). This high productivity is associated with large water requirements, as also shown by the high gL of well-watered plants, exceeding 600 mmol s−1 m−2. Drought stress caused gL to decline by about 50% (Figure 1). This confirms that, despite some degree of genotype variability, poplar is overall very sensitive to drought (Chen et al. 1997, Brignolas et al. 2000, Lambs and Muller 2002) even when it is moderate like in our experimental conditions (Centritto et al. 2011b, Marino et al. 2017), as indicated by the slight decrease in ΨLM (~−1 MPa, Figure 2). Drought stress also induced a significant reduction of maximum PSII efficiency (Figure 1). However, after overnight relief of water stress, pre-stress values of Fv/Fm were fully restored despite the fact that gL values remained low. These results indicate that there was no permanent damage in the reaction centres of PSII in WS leaves, and the drop in Fv/Fm was a likely consequence of transient photoinhibitory processes, leading to a lower ability of leaves to dissipate the excess excitation energy (Yin et al. 2006). Many reports have shown that poplar displays a high vulnerability to xylem embolism (Hukin et al. 2005, Awad et al. 2010, Secchi and Zwiniecki 2012, Barigah et al. 2013, Barigah et al. 2013, Fichot et al. 2015, Schreiber et al. 2016). Moreover, many Populus species display a high native level of embolism during the growing season, with a pool of permanently non-functional xylem conduits building up PLC frequently over 40% even in well-watered plants (Secchi and Zwieniecki 2012). Consistent with these observations, in our 2-year-old P. nigra stem segments, which contained xylem formed in the current and the previous year, we found a PLC of about 40% in WW plants (Figure 3). Furthermore, water stress resulted in a significant reduction in gL (about 50%) and in Kstem (about 60%). The slight decrease of Ψx and ΨLM was accompanied by a marked increase of PLC (>60%), indicating a typical near-isohydric behaviour of this genotype characterized by high vulnerability to embolism (Awad et al. 2010, Schreiber et al. 2016). Xylem vulnerability curves of Populus nigra (Secchi and Zwieniecki 2012) are very steep, in the range between −0.6 and −1 MPa, so that even minor differences in water potential can result in significant changes in PLC values. We suggest that this is exactly the case in our study, where even a not significant drop in water potential between WW and WS plants corresponded to a significant increase in PLC values. Stomatal closure efficiently buffered the drop of Ψx and ΨLM under water-stress conditions, confirming previous observations (Secchi and Zwieniecki 2012). Stomatal closure during moderate water-stress is likely supported by ABA released in the xylem sap and transported to leaves. This was apparently the case in our study (Figure 4), with plants exposed to moderate stress that underwent stomatal closure accompanied by a significant increase of free ABA in the xylem sap (Li et al. 2011; Romero et al. 2012; Secchi et al. 2013). It is important to mention that ABA produced in response to water stress originates in leaves, and most of the ABA stored in the roots is translocated via phloem from leaves to roots, as previously hypothesized by Holbrook et al. (2002) and Christmann et al. (2005) and recently confirmed by compelling evidence (Manzi et al. 2015, McAdam et al. 2016, Mitchell et al. 2016). Early stomatal closure reduces the risk of xylem cavitation and avoids hydraulic dysfunction, but also decreases photosynthetic rates thus reducing carbon gain (Tyree and Sperry 1988, Choat et al. 2012, Manzoni 2014, Yi et al. 2017). Soluble carbohydrates are involved in multiple processes to alleviate the adverse effects of drought stress (Keunen et al. 2013). In particular, in the post-drought recovery of embolism, NSC likely constitute the main components of the osmotic force required to drive water flow into embolized conduits, thus sustaining the hydraulic recovery process (Hacke and Sperry 2003, Nardini et al. 2011, Brodersen and McElrone 2013). In particular, embolism could trigger a drop in starch content associated with changes in gene expression levels of α- and β-amylases (Secchi and Zwieniecki 2011) and with a consequent release of sucrose from parenchyma cells to non-functional conduits (Regier et al. 2009, Salleo et al. 2009, Secchi and Zwieniecki 2010). In our experiment, the imposed drought stress did not alter the pool of stem soluble NSC in wood or bark of WW and WS plants, with total NSC averaging 140 mg g−1 DW (Figure 5). This suggests that moderate water stress does not impact the carbon status of the poplar genotype under study. Indeed, it has been previously reported that severe drought can lead to little or no changes in NSC pools (Anderegg and Anderegg 2013; Adams et al. 2017; Tomasella et al. 2017). However, we recorded an increase in soluble NSC at the recovery stage, and a parallel decrease by about 35% of starch reserves stored in the bark (Figure 5), possibly suggesting rapid mobilization to fuel the hydraulic recovery process (about 30 mg g−1 DW). This result is consistent with recently reported observations by Tomasella et al. (2017), where drought-induced PLC in saplings of Norway spruce was fully recovered after re-irrigation, paralleled by starch conversion into soluble sugars. According to previous reports, the content of stem NSC recorded in our plants was sufficient to build up the osmotic forces required to recover the water transport capacity (Figure 3). In fact, on the basis of some over-simplified calculations (Nardini et al. 2017), the theoretical amount of glucose required to osmotically reverse xylem embolism (5.7 mg g−1 DW) would correspond to only about 4% or 37% of total NSC or soluble stem NSC at the peak of the drought, respectively. A similar theoretically low amount of total NSC (7%) was recently reported to be sufficient for Ailanthus altissima saplings to completely recover from a PLC of about 80% after stress relief (Savi et al. 2016a; Nardini et al. 2017). It should be noted that these rough calculations represent simplistic estimates, since it is not known which fraction of NSC reserves can be actually mobilized and converted into glucose to fuel hydraulic recovery. In addition, we cannot exclude further important roles of carbohydrate mobilization during the drought recovery, when stomatal conductance was still limited, such as the utilization of sugars as possible substrates for respiration, the synthesis of other molecules and the maintenance of secondary growth and stem elongation (Hartmann and Trumbore 2016, Traversari et al. 2018). Our data further suggest that NSC used for hydraulic recovery derive from mobilization of starch reserves stored in the bark (Figure 7). Interestingly, at the end of the drought period we did not observe any ABA change either in the wood or in the bark, but a significant increase in the xylem sap (Figure 4). After re-watering, ABA content peaked in xylem sap and in both wood and bark, suggesting divergent roles for ABA in different plant organs/tissues. It is noteworthy that, following re-watering, the content of free ABA increased overnight about ten-fold compared with water-stressed plants. We speculate that the overnight re-equilibrium in the xylem flow from the root to the shoot, as shown by the recovery in ΨLM in the re-irrigated plants, may have resulted in a sudden release of root-stored free ABA with consequent dramatic build-up of this hormone in the xylem sap. This finding also explains the persistent low gL and Kstem in the re-irrigated plants despite the recovery of ΨLM (Holbrook et al. 2001, Lovisolo et al. 2008, Tombesi et al. 2015). Indeed, increased concentration of ABA in xylem sap may reduce not only gL but also hydraulic conductance (Pantin et al. 2013, Coupel-Ledru et al. 2017). Different from previous results in Vitis vinifera, where stomatal conductance remained low upon rewatering mainly because of hydraulic signals caused by embolism (Pou et al. 2008), the strict hormonal control on gL and Kstem observed in poplar under restored well-watered conditions could be beneficial, since water loss limitation by transpiration rate might help reducing residual xylem tension during the process of hydraulic recovery (Tombesi et al. 2015). Our results suggest novel and important functions of free-ABA in the modulation of NSC metabolism in the stem of RW plants. In fact, a clear relationship emerged between free-ABA and the concentration of soluble sugars (but not of starch) in wood (Figure 6 and 7), suggesting a possible role for this hormone in restoring xylem hydraulic integrity through NSC mobilization. At the same time, both the observed positive relationship between free-ABA and monosaccharides as well as the negative relationship between free-ABA and starch found in the bark apparently suggest that starch degradation is mediated by this hormone, with a consequent accumulation of soluble sugars in the stem tissues. Hence, we surmise a possible indirect role of ABA in restoring the sugar content in wood during the hydraulic recovery, thus decreasing the short-term risk of carbon starvation. The sharp increment in free-ABA content observed in wood and in bark upon rewatering was accompanied by similar trends of PA and DPA catabolites, and this accumulation was probably due to a de novo synthesis rather than a de-glycosylation of ABA-GE (Endo et al. 2008). In particular, the significant activation of ABA catabolism can restrict excessive ABA accumulation, thus controlling the homeostasis of this hormone in different tissues (Ren et al. 2007). Recently, it has been shown that PA can have biological activity similar to free ABA, since it interacts with the same ABA receptors (Weng et al. 2016). Overall, our results are in line with previous studies showing the interaction and coordination between wood and bark (Hölttä et al. 2006, Mencuccini et al. 2013, Diaz-Espejo and Hernandez-Santana 2017), and suggest the putative involvement of the phloem in embolism repair processes. There is evidence for hydraulic connections between xylem and phloem (Steppe et al. 2006, Sevanto et al. 2011, Mencuccini et al. 2013), and that phloem provides solutes or water, or even both, to sustain the osmotic mechanisms of hydraulic recovery (‘phloem-unloading’ hypothesis, Salleo et al. 2004, 2009). Furthermore, recent theoretical calculations have demonstrated that simultaneous adjustments of water and sugar fluxes between these two tissues are strictly linked to soil water uptake as well as stomatal regulation (Hölttä et al. 2017). In addition, a quick hydraulic recovery process mediated by osmotic pressure after re-watering has been previously observed (Brodersen et al. 2010, Trifilò et al. 2014, 2019) and also suggested by model calculations, which estimated a refilling time in the order of 10 h even under the condition of water tension in the xylem (Vesala et al. 2003). Our data suggest that phloem-unloading processes could even take place during fast hydraulic recovery upon restoration of well-watered conditions, as while total NSC content did not change in stem of water-stressed plants, starch in bark was significantly reduced after re-watering. This change was accompanied by an increase in disaccharides and monosaccharides (in particular fructose) in both wood and bark tissues. Therefore, it is likely that restoration of xylem functionality is facilitated when water availability increases after a drought spell, and ABA may have a prominent role in the mobilization of soluble carbohydrates from the bark, thus preventing the risk of wood carbon store depletion after embolism repair. However, rapid hydraulic recovery of embolized vessels may not be a common mechanism in tree species. Indeed, other experiments have shown that embolism formed during severe drought events persist, leaving some level of dysfunction in the hydraulic system (Choat et al. 2018, Creek et al. 2018), suggesting that the restoration of the hydraulic function can be achieved only when corticular photosynthesis takes place (Liu et al. 2019), or under root/stem pressure (Yang et al. 2012, Knipfer et al. 2015) or after the production of new xylem vessels (Hacke and Sauter 1996). In summary, in the poplar genotype tested in our study the capacity to control stomatal opening and reallocate carbohydrates after water stress could improve the recovery of xylem functionality after the loss of stem conductivity. Our study suggests a possible involvement of ABA in the process of hydraulic recovery, but further studies will be required for incorporating mechanistic evidences on how sugar and ABA regulatory pathways are integrated both in bark and wood under different water-deficit conditions. Authors' contributions C.B., A.N. and M.C. conceived the experiment; C.B. and T.S. performed the experiment and analyzed the samples; all the authors contributed to writing and revising the manuscript. Acknowledgments This research was supported by the EU-FP7 project WATBIO (Development of improved perennial non-food biomass and bioproduct crops for water stressed environments—no.311929). References Adams HD , Zeppel MJ, Anderegg WR et al. 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TI - Changes in abscisic acid content during and after drought are related to carbohydrate mobilization and hydraulic recovery in poplar stems
JF - Tree Physiology
DO - 10.1093/treephys/tpaa032
DA - 2020-07-30
UR - https://www.deepdyve.com/lp/oxford-university-press/changes-in-abscisic-acid-content-during-and-after-drought-are-related-foSnSt7UGj
SP - 1043
EP - 1057
VL - 40
IS - 8
DP - DeepDyve
ER -